In the year 2000, an estimated 22 million people were suffering from cancer worldwide and 6.2 millions deaths were attributed to this class of diseases. Every year, there are over 10 million new cases and this estimate is expected to grow by 50% over the next 15 years (WHO, World Cancer Report. Bernard W. Stewart and Paul Kleihues, eds. IARC Press, Lyon, 2003). Current cancer treatments are limited to invasive surgery, radiation therapy and chemotherapy, all of which cause either potentially severe side-effects, non-specific toxicity and/or traumatizing changes to ones body image and/or quality of life. Cancer can become refractory to chemotherapy reducing further treatment options and likelihood of success. The prognosis for some cancer is worse than for others and some, like lung or pancreatic cancer are almost always fatal. In addition, some cancers with a relatively high treatment success rate, such as breast cancer, also have a very high incidence rate and, thus, remain major killers.
For instance, there are over 1 million new cases of breast cancer, worldwide, each year. Treatments consist of minimal to radical surgical removal of breast tissue and lymph nodes with radiation and chemotherapy for metastatic disease. Prognosis for localized disease is relatively good with a 5 years survival rate of around 50% but once the cancer has metastasized, it is incurable with an average survival of around 2 years. Despite improving treatment success rates, nearly 400,000 women die of breast cancer each year, the highest number of deaths to cancer in woman, ahead of deaths to lung cancer. Among the short and long term survivors, most will suffer the lifelong trauma of the invasive and disfiguring surgical treatment.
Another example is liver cancer, with more than half a million new cases each year and nearly the same number of deaths due to poor treatment efficacy. Hepatocellular carcinomas represent around 80% of all liver cancers and are rarely curable. Five-year survival rate is only about 10% and survival after diagnosis often less than 6 months. Although surgical resection of diseased tissue can be effective, it is not an option for the majority of cases because of the presence of cirrhosis of the liver. Hepatocellular carcinomas are largely radiation resistant and response to chemotherapy is poor.
Yet another example is that of pancreatic cancer with around 200,000 new cases per year and a very poor prognosis. In fact, the majority of patients die within a year of diagnosis and only a few percent of patients survive five years. Surgery is the only available treatment but is associated with high morbidity and complication rates because it involves not only the resection of at least part of the pancreas, but also of all of the duodenum, part of the jejunum, bile duct and gallbladder and a distal gastrectomy. In some cases, the spleen and lymph nodes are also removed.
Bladder cancer is the 9th most common cancer worldwide with an estimated 330,000 new cases and 130,000 deaths each year. In Europe, this disease is the cause of death for approximately 50,000 people each year. Current treatment includes the intravesicular delivery of chemotherapy and immunotherapy with the bacille Calmette-Guerin (BCG) vaccine that involves the additional risk of systemic infection with the tuberculosis bacterium. Despite this aggressive treatment regime, 70% of these superficial papillary tumors will recur over a prolonged clinical course some will progress into invasive carcinomas. The high rate of recurrence of this disease and associated repeated course of treatment makes this form of cancer one of the most expensive to treat over a patient's lifetime. For patients with recurring disease, the only options are to undergo multiple anesthetic-requiring cystoscopy surgery or major, radical, life-altering surgery (usually cystectomy). Radical cystectomy consists of excision of the bladder, prostate and seminal vesicle in males and of the ovaries, uterus, urethra and part of the vagina in females.
There are many more examples of cancer where current treatments do not meet the needs of patients either due to their lack of efficacy and/or because they have high morbidity rates and severe side-effects. Those selected statistics and facts however, illustrate well the need for cancer treatments with better safety and efficacy profiles.
One of the causes for the inadequacy of current cancer treatments is their lack of selectivity for affected tissues and cells. Surgical resection always involves the removal of apparently normal tissue as a “safety margin” which can increase morbidity and risk of complications. It also always removes some of the healthy tissue that may be interspersed with tumor cells and that could potentially maintain or restore the function of the affected organ or tissue. Radiation and chemotherapy will kill or damage many normal cells due to their non-specific mode of action. This can result in serious side-effects such as severe nausea, weight loss and reduced stamina, loss of hair etc., as well as increasing the risk of developing secondary cancer later in life. Treatment with greater selectivity for cancer cells would leave normal cells unharmed thus improving outcome, side-effect profile and quality of life.
The selectivity of cancer treatment can be improved by using antibodies that are specific for molecules present only or mostly on cancer cells. Such antibodies can be used to modulate the immune system and enhance the recognition and destruction of the cancer by the patient's own immune system. They can also block or alter the function of the target molecule and, thus, of the cancer cells. They can also be used to target drugs, genes, toxins or other medically relevant molecules to the cancer cells. Such antibody-drug complexes are usually referred to as immunotoxins or immunoconjugates and a number of such compounds have been tested in recent year [Kreitman R J (1999) Immunotoxins in cancer therapy. Curr Opin Immunol 11:570-578; Kreitman R J (2000) Immunotoxins. Expert Opin Pharmacother 1:1117-1129; Wahl R L (1994) Experimental radioimmunotherapy. A brief overview. Cancer 73:989-992; Grossbard M L, Fidias P (1995) Prospects for immunotoxin therapy of non-Hodgkin's lymphoma. Clin Immunol Immunopathol 76:107-114; Jurcic J G, Caron P C, Scheinberg D A (1995) Monoclonal antibody therapy of leukemia and lymphoma. Adv Pharmacol 33:287-314; Lewis J P, DeNardo G L, DeNardo S J (1995) Radioimmunotherapy of lymphoma: a UC Davis experience. Hybridoma 14:115-120; Uckun F M, Reaman G H (1995) Immunotoxins for treatment of leukemia and lymphoma. Leuk Lymphoma 18:195-201; Kreitman R J, Wilson W H, Bergeron K, Raggio M, Stetler-Stevenson M, FitzGerald D J, Pastan I (2001) Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. N Engl J Med 345:241-247]. Most antibodies tested to date have been raised against known cancer markers in the form of mouse monoclonal antibodies, sometimes “humanized” through molecular engineering. Unfortunately, their targets can also be present in significant quantities on a subset of normal cells thus raising the risk of non-specific toxic effects. Furthermore, these antibodies are basically mouse proteins that are being seen by the human patient's immune system as foreign proteins. The ensuing immune reaction and antibody response can result in a loss of efficacy or in side-effects.
The inventors have used a different approach in their development of antibodies for cancer treatment. Instead of immunizing experimental animals with cancer cells or isolated cancer cell markers, they have sought out only those markers that are recognized by the patient's own immune system or, in other words, that are seen by the immune system as a foreign molecule. This implies that the markers or antigens are usually substantially absent on normal cells and, thus, the risk of non-specific toxicity is further reduced. Hybridoma libraries are generated from cancer patient-derived lymphocytes and the antibodies they secrete are tested for binding to normal and tumor cells. Only antibodies showing high selectivity for cancer cells are retained for further evaluation and development as a cancer therapeutic or diagnostic agent. One such highly selective antibody is the subject of this patent application. In addition to being selective, this antibody is fully compatible with the patient's immune system by virtue of being a fully-human protein. The antibody of the invention can be used for diagnostic or therapeutic uses or as a basis for engineering other binding molecules for the target antigen.
The basic structure of an antibody molecule consists of four protein chains, two heavy chains and two light chains. These chains are interconnected by disulfide bonds. Each light chain is comprised of a light chain variable region and a light chain constant region. Each heavy chain is comprised of a heavy chain variable region and a heavy chain constant region. The light chain and heavy chain variable regions can be further subdivided into framework regions and regions of hypervariability, termed complementarity determining regions (CDR). Each light chain and heavy chain variable region is composed of three CDRs and four framework regions.
CD44 represents a family of cell surface glycoproteins encoded by a single gene comprising a total of 20 exons. Exons 19 and 20 are expressed together as the cytoplasmic tail and therefore grouped as “exon 19” by most research groups (Liao et al. J. Immunol. 151:6490-99, 1993). The term exon 19 will be used henceforth to designate genomic exons 19 and 20. Structural and functional diversity is achieved by alternative splicing of the messenger RNA involving 10 “variant” exons identified as exons 6-15 or, most often, as “variant exons” 1-10 (v1-v10). In human, variant exon 1 contains a stop codon and is not usually expressed. The longest potential CD44 variant is therefore CD44v2-10 (see Naor et al. Adv Cancer Res 71:241-319, 1997 for review of CD44).
Exons 1-5 and all variant exons are part of the extracellular domain and contain many potential sites for post-translational modifications. The transmembrane domain is highly conserved across species but the intracellular tail can be truncated leading to another type of variant. One such variant comprises variant exons 8-10 but lacks part of exon 19. Changes to the intracellular domain has been shown to change the function of CD44, in part with respect to binding and internalization of hyaluronic acid (HA). CD44 is not only involved in binding to the extracellular molecules but it also has cell signaling properties (see Turley et al. J Biol Chem 277(7):4589-4592, 2002 for review).
The “standard” CD44 (CD44s), the most commonly expressed form of CD44, contains exons 1-5 and 16-19 and none of the variant exons. The molecular weight for the core protein is 37-38 kDa but posttranslational modification can result in a molecule of 85-95 kDa or more (Drillenburg et al., Blood 95(6): 1900, 2000). It binds hyaluronic acid (HA), an extracellular glycosaminoglycan, constitutively and CD44 is often referred to as the HA receptor. It is interesting that the presence of variant exons can reduce the binding of HA by CD44 such that CD44 variants cannot be said to constitutively bind HA but such binding can be inducible (reviewed in Naor et al. Adv Cancer Res 71:241-319, 1997). See FIG. 17 for some examples of variants.
CD44E, also called CD44v8-10, contains variant exons 8-10 in addition to the exons 1-5 and 16-19. Other variants include CD44v3-10, CD44v6, CD44v7-8 and many others. The variant exons are part of the extracellular domain of the CD44.
CD44E can be present on certain normal epithelial cells, particularly by generative cells of the basal cell of stratified squamous epithelium and of glandular epithelium (Mackay et al. J Cell Biol 124(1-2):71-82, 1994) and in the fetus at certain stages development. But importantly, it has been shown to be overexpressed on various types of cancer cells. Using RT-PCR, lida & Bourguignon (J Cell Physiol 162(1):127-133, 1995) and Kalish et al. (Frontiers Bioscience 4(a):1-8, 1999) have shown that CD44E is present in normal breast tissue and is more abundant than CD44s. They have also shown that CD44, including CD44E and CD44s are overexpressed, and preferentially located in metastatic breast cancer tissues. Miyake et al. (J Urol 167(3):1282-87, 2002) reported that CD44v8-10 mRNA is strongly expressed in urothelial cancer and can even be detected in urinary exfoliated cells of patients with invasive vs superficial urothelial cancer. The ratio of CD44v8-10 to CD44v10 mRNA increases in cancer and was shown to have diagnostic value in breast, lung, laryngeal and bladder. The presence of CD44v8-10 was also confirmed by immunohistochemistry with a polyclonal antibody (Okamoto et al. J Natl Cancer Inst 90(4): 307-15, 1997). CD44v8-10 can also be overexpressed in gallbladder cancer (Yamaguchi et al. Oncol Rep 7(3):541-4, 2000), renal cell carcinoma (Hara et al. Urology 54(3):562-6, 1999), testicular germ cell tumors (Miyake et al. Am J Pathol 152(5): 1157-60, 1998), non-small cell lung carcinomas (Sasaki et al. Int J Oncol 12(3):525-33, 1998), colorectal cancer (Yamaguchi et al. J Clin Oncol 14(4): 1122-27, 1996) and gastric cancer (Yamaguchi et al. Jpn J Cancer Res 86(12): 1166-71, 1995). Overexpression of CD44v8-10 was also shown to have diagnostic value for prostate cancer (Martegani et al. Amer J Pathol 154(1): 291-300, 1999).
Alpha-fetoprotein (AFP) is a major serum protein synthesized during fetal life. Its presence in adults is usually indicative of carcinomas, particularly those of the liver and teratocarcinomas. It is part of the albuminoid gene family that also comprises serum and alpha albumins and vitamin D-binding protein. AFP comprises 590 amino acids for a molecular weight of about 69-70 kDa and has one site for glycosylation. (Morinaga et al., Proc Natl Acad Sci 80:4604-08, 1983; Mizejewski Exp Biol Med 226(5):377-408, 2002). Molecular variants have been studied and identified in rodents, but in humans there are no reports of variant proteins being detected. A recent report has identified a variant mRNA that, if expressed, would code for a 65 kDa protein. This protein is expected to remain in the cytoplasm (Fukusawa et al. J Soc Gynecol Investig May 20, e-publication, 2005).